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Search Results: 1 - 10 of 146033 matches for " Janet F Partridge "
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Correcting for sequence biases in present/absent calls
Eugene F Schuster, Eric Blanc, Linda Partridge, Janet M Thornton
Genome Biology , 2007, DOI: 10.1186/gb-2007-8-6-r125
Abstract: The Affymetrix GeneChip technology uses a simple method to distinguish 'true' biological signal from background noise. Labeled cRNA transcripts are hybridized to 25 base-pair (bp) oligonucleotide 'probes' covalently bound to the array. Probes are designed in pairs with one probe designed to perfectly match the target transcript (PM probe) and the other designed to measure the non-specific binding signal of its partner PM probe. The mismatch (MM) probe is identical to its partner PM probe except for the central (13th) nucleotide, which is changed to the complementary base. Ideally, the subtraction of MM probe signal from its partner PM probe signal results in the removal of non-specific background and a target transcript specific signal. To gain a more robust target transcript signal, there are typically 11-20 PM-MM probe-pairs within a probeset that query different sequences of the same target transcript. The probeset design also allowed Liu et al. [1] to create an algorithm (the MAS 5.0 algorithm) that detects the presence or absence of a target transcript.PM probe signals that are greater than their partner MM probe signals imply that the target transcripts are present. If the PM signal is equal to its partner MM signal, then it is likely that both PM and MM probes report non-specific binding and the target transcript is absent from the labeled cRNA. For a probe-pair, a discrimination score R can be calculated by (PM - MM)/(PM + MM). Liu and colleagues' MAS 5.0 detection algorithm is based on the distribution of R scores for every probe-pair within a probeset. The classification of presence or absence is based on P values generated with the one-sided Wilcoxon signed rank test [2]. The benefits of the Wilcoxon signed rank test is that it is a non-parametric test, insensitive to outliers and there are well established methods to generate confidence levels (that is, P values) [3].For the present/absent algorithm, the null hypothesis in the Wilcoxon signed rank test i
Estimation and correction of non-specific binding in a large-scale spike-in experiment
Eugene F Schuster, Eric Blanc, Linda Partridge, Janet M Thornton
Genome Biology , 2007, DOI: 10.1186/gb-2007-8-6-r126
Abstract: We have found that the MAS5 perfect match-mismatch (PM-MM) model is a poor model for estimation of NSB, and that the Naef and Zhang sequence-based models can reasonably estimate NSB. In general, using the GC robust multi-array average, which uses Naef binding affinities, to calculate NSB (GC-NSB) outperforms other methods for detecting differential expression. However, there is an intensity dependence of the best performing methods for generating probeset expression values. At low intensity, methods using GC-NSB outperform other methods, but at medium intensity, MAS5 PM-MM methods perform best, and at high intensity, MAS5 PM-MM and Zhang's position-dependent nearest-neighbor (PDNN) methods perform best.A combined statistical analysis using the MAS5 PM-MM, GC-NSB and PDNN methods to generate probeset values results in an improved ability to detect differential expression and estimates of false discovery rates compared with the individual methods. Additional improvements in detecting differential expression can be achieved by a strict elimination of empty probesets before normalization. However, there are still large gaps in our understanding of the Affymetrix GeneChip technology, and additional large-scale datasets, in which the concentration of each transcript is known, need to be produced before better models of specific binding can be created.Despite the ubiquitous use of Affymetrix GeneChip arrays (Affymetrix has recorded more than 3,600 publications with data collected on this platform), we have a limited understanding of the technology. The physico-chemical details of hybridization of target mRNA on these arrays are still incomplete and models for specific and non-specific DNA-RNA interactions are continuously being refined (a recent example can be found in [1]). A deeper understanding of these processes is required to better separate experimental variation from biological variation. For example, it would allow for addressing the influence of the amount of labe
Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex
Sreenath Shanker equal contributor,Godwin Job equal contributor,Olivia L. George,Kevin M. Creamer,Alaa Shaban,Janet F. Partridge
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1001174
Abstract: Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent “priRNAs.” The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3WG) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi–independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4+ in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi–independent factors in the assembly of heterochromatin.
Genetic Effects at Pleiotropic Loci Are Context-Dependent with Consequences for the Maintenance of Genetic Variation in Populations
Heather A. Lawson ,Janet E. Cady,Charlyn Partridge,Jason B. Wolf,Clay F. Semenkovich,James M. Cheverud
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002256
Abstract: Context-dependent genetic effects, including genotype-by-environment and genotype-by-sex interactions, are a potential mechanism by which genetic variation of complex traits is maintained in populations. Pleiotropic genetic effects are also thought to play an important role in evolution, reflecting functional and developmental relationships among traits. We examine context-dependent genetic effects at pleiotropic loci associated with normal variation in multiple metabolic syndrome (MetS) components (obesity, dyslipidemia, and diabetes-related traits). MetS prevalence is increasing in Western societies and, while environmental in origin, presents substantial variation in individual response. We identify 23 pleiotropic MetS quantitative trait loci (QTL) in an F16 advanced intercross between the LG/J and SM/J inbred mouse strains (Wustl:LG,SM-G16; n = 1002). Half of each family was fed a high-fat diet and half fed a low-fat diet; and additive, dominance, and parent-of-origin imprinting genotypic effects were examined in animals partitioned into sex, diet, and sex-by-diet cohorts. We examine the context-dependency of the underlying additive, dominance, and imprinting genetic effects of the traits associated with these pleiotropic QTL. Further, we examine sequence polymorphisms (SNPs) between LG/J and SM/J as well as differential expression of positional candidate genes in these regions. We show that genetic associations are different in different sex, diet, and sex-by-diet settings. We also show that over- or underdominance and ecological cross-over interactions for single phenotypes may not be common, however multidimensional synthetic phenotypes at loci with pleiotropic effects can produce situations that favor the maintenance of genetic variation in populations. Our findings have important implications for evolution and the notion of personalized medicine.
H3K9me-Independent Gene Silencing in Fission Yeast Heterochromatin by Clr5 and Histone Deacetylases
Klavs R. Hansen,Idit Hazan,Sreenath Shanker,Stephen Watt,Janne Verhein-Hansen,Jürg B?hler,Robert A. Martienssen,Janet F. Partridge,Amikam Cohen,Geneviève Thon
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1001268
Abstract: Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.
Plasticity of Fission Yeast CENP-A Chromatin Driven by Relative Levels of Histone H3 and H4
Araceli G Castillo equal contributor,Barbara G Mellone equal contributor,Janet F Partridge,William Richardson,Georgina L Hamilton,Robin C Allshire ,Alison L Pidoux
PLOS Genetics , 2007, DOI: 10.1371/journal.pgen.0030121
Abstract: The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-ACnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-ACnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-ACnp1 chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-ACnp1 associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-ACnp1 for assembly into central domain chromatin, resulting in less CENP-ACnp1 and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-ACnp1 influence the extent of DNA at centromeres that is packaged in CENP-ACnp1 chromatin and the composition of this chromatin. Thus, CENP-ACnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-ACnp1 and other core histones.
Using Answer Set Programming to Integrate RNA Expression with Signalling Pathway Information to Infer How Mutations Affect Ageing
Irene Papatheodorou, Matthias Ziehm, Daniela Wieser, Nazif Alic, Linda Partridge, Janet M. Thornton
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0050881
Abstract: A challenge of systems biology is to integrate incomplete knowledge on pathways with existing experimental data sets and relate these to measured phenotypes. Research on ageing often generates such incomplete data, creating difficulties in integrating RNA expression with information about biological processes and the phenotypes of ageing, including longevity. Here, we develop a logic-based method that employs Answer Set Programming, and use it to infer signalling effects of genetic perturbations, based on a model of the insulin signalling pathway. We apply our method to RNA expression data from Drosophila mutants in the insulin pathway that alter lifespan, in a foxo dependent fashion. We use this information to deduce how the pathway influences lifespan in the mutant animals. We also develop a method for inferring the largest common sub-paths within each of our signalling predictions. Our comparisons reveal consistent homeostatic mechanisms across both long- and short-lived mutants. The transcriptional changes observed in each mutation usually provide negative feedback to signalling predicted for that mutation. We also identify an S6K-mediated feedback in two long-lived mutants that suggests a crosstalk between these pathways in mutants of the insulin pathway, in vivo. By formulating the problem as a logic-based theory in a qualitative fashion, we are able to use the efficient search facilities of Answer Set Programming, allowing us to explore larger pathways, combine molecular changes with pathways and phenotype and infer effects on signalling in in vivo, whole-organism, mutants, where direct signalling stimulation assays are difficult to perform. Our methods are available in the web-service NetEffects: http://www.ebi.ac.uk/thornton-srv/softwa?re/NetEffects.
The chicken or the egg? Investigating the transformational impact of learning technology
Janet F. Buchan
Research in Learning Technology , 2011, DOI: 10.3402/rlt.v19i2.10355
Abstract: This study aimed to investigate the transformational impact of introducing significant new learning technology in an Australian university over the time period 2007–2009. The exploration of this transformation is grounded in a social–ecological systems approach to the management of technology enhanced learning environments in the face of constant change. The transformational impact is described using the Adaptive Cycle Framework. The single case study had a whole-of-institution systems focus. Data collection targeted a variety of stakeholders involved in the use of, support for users of, implementation and maintenance of learning technology. A variety of data collection methods were used including interviews, document collection and a reflective journal. The research shows that learning technology has significant transformational potential at both individual and at institutional levels. However, this study has unearthed a ‘chicken or the egg' conundrum. Although learning technology is an important part of the future of educational institutions, the adaptability of the organisation and capacity to predict, plan for and support ongoing changes in learning technology is an important part of realising the transformational potential and effectiveness of learning technology.
Why is spirometry underused in the diagnosis of the breathless patient: a qualitative study
Nicola J Roberts, Susan F Smith, Martyn R Partridge
BMC Pulmonary Medicine , 2011, DOI: 10.1186/1471-2466-11-37
Abstract: Five separate focus groups were undertaken with final year medical undergraduates, junior hospital doctors, general practitioners (GPs) and specialist trainees in respiratory medicine. The participants were not told prior to the session that we were specifically interested in their views about spirometry but discussion was moderated to elicit their approaches to the diagnosis of a breathless patient, their use of investigations and their learning preferences.Undergraduates and junior doctors rarely had a systematic approach towards the breathless patient and tended, unless prompted, to focus on the emergency room situation rather than on patients with longer term causes of breathlessness. Whilst their theoretical knowledge embraced the possibility of a non-respiratory cause for breathlessness, neither undergraduates nor junior doctors spontaneously mentioned the use of spirometry in the diagnosis of respiratory disease. When prompted they cited lack of familiarity with the use and location of equipment, and lack of encouragement to use it as being major barriers to utilization. In contrast, GPs and specialist respiratory trainees were enthusiastic about its use and perceived spirometry as a core element of the diagnostic workup.More explicit training is needed regarding the role of spirometry in the diagnosis and management of those with lung disease and this necessitates both practical experience and training in interpretation of the data. However, formal teaching is likely to be undermined in practice, if the concept is not strongly promoted by the senior staff who act as role models and trainers.There are over 40 common respiratory conditions many of which share symptoms with disorders of other systems. Breathlessness for example may be due to heart or lung disease, diaphragm weakness, pulmonary vascular disease or systemic disorders such as anaemia, obesity or hyperthyroidism. The correct differentiation requires a systematic approach which may develop with expe
Comparison of a web-based package with tutor-based methods of teaching respiratory medicine: subjective and objective evaluations
Susan F Smith, Nicola J Roberts, Martyn R Partridge
BMC Medical Education , 2007, DOI: 10.1186/1472-6920-7-41
Abstract: 137 out of 315 final year undergraduate students in a single medical school volunteered to take part. Each received up to two hours of tutor-lead interactive, tutor-lead didactic or electronic, Web-based teaching on the accurate diagnosis and management of respiratory disease. Post teaching performance was assessed by multiple true/false questions and data interpretation exercises, whilst students' teaching preferences were assessed by questionnaire.Despite a high knowledge baseline before the study, there was a small, but statistically significant increase in knowledge score after all forms of teaching. Similarly, data interpretation skills improved in all groups, irrespective of teaching format, Although paradoxically most students expressed a preference for interactive tutor-lead teaching, spirometry interpretation in those receiving web-based teaching improved significantly more [p = 0.041] than in those in the interactive group.Web-based teaching is at least as good as other teaching formats, but we need to overcome students' reluctance to engage with this teaching method.The United Kingdom, like many other countries, has a shortage of doctors. As a result, there has been a significant recent increase in the number of students entering medical school. Over the same time there has been a reduction in appointment of academic clinicians [1-3], an increase in pressure on clinical staff to deliver demanding service targets and the introduction of legislation limiting hours in the workplace. Together these factors have combined to reduce the number of staff available to teach. An additional problem for teachers of respiratory medicine is that many respiratory patients are treated within the community, rather than as hospital in-patients, thus potentially limiting the range of direct experience open to undergraduate and junior postgraduate trainees. Another significant pressure at present is the need in the United Kingdom to develop a coherent programme of postgraduat
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